Acteoside in Chinese Natural Plant-Cistanche To Ameliorate Kidney Disease--Part I
Mar 03, 2022
Contact: emily.li@wecistanche.com
A natural product of acteoside ameliorate kidney injury in diabetes db/db mice and HK-2 cells via regulating NADPH/ oxidase-TGF-β/Smad signaling pathway
Qinwen Wang | Xinxin Dai et.al
This study was designed to investigate the protective effects and mechanisms of acteoside on DKD in diabetes male db/db mice and high glucose-induced HK-2 cells. The diabetes db/db mice were divided randomly into the model group, metformin group, irbesartan group, and acteoside group. We observed the natural product of acteoside exhibiting a significant effect in renal protection through analysis of biochemical indicators and endogenous metabolites, histopathological observations, and western blotting. HK-2 cells subjected to high glucose were used in in-vitro experiments. The molecular mechanisms were investigated by RT-PCR and western blot. Acteoside prevents high glucose-induced HK-2 cells and diabetes db/db mice by inhibiting NADPH/oxidase-TGF-β/Smad signaling pathway. Acteoside regulated the disturbed metabolic pathway of lipid metabolism, glyoxylate and dicarboxylate metabolism, and arachidonic acid metabolism. We discovered the natural product of acteoside exhibits a significant effect on renal protection. This study paved the way for further exploration of pathogenesis, early diagnosis, and the development of a new therapeutic agent for DKD.
KEYWORDS acteoside, diabetic kidney disease, metabolic profiling, NADPH/oxidase-TGF-β/Smad signaling pathway, ROS
Part I
Acteoside treat Kidney Disease
1 | INTRODUCTION
Acteoside is a representative component of phenylethanoid glycosides, widely distributed in 79 genera of plants, such as Rehmannia glutinosa, Cistanche deserticola, and other medicinal plants. Acteoside is characterized by caffeic acid and hydroxytyrosol bound to a glucose moiety through ester and glycosidic bonds, respectively, with a rhamnose unit linked to the glucose molecule (Zhao et al., 2015). It is reported that acteoside possesses widely pharmacological activities, such as kidney protection (Gan et al., 2018), antioxidation (He et al., 2011), neuroprotection (Li, Zhou, Xu, Song, & Lu, 2018), liver protection (Lee et al., 2004), anticancer (Ohno, Inoue, Ogihara, & Saracoglu, 2002), and so on. However, the protective effects of acteoside in diabetic renal injury are unclear.
Diabetes is one of the fastest-growing global health emergencies of the 21st century. In 2019, it is estimated that 463 million people have diabetes and this number is projected to reach 578 million by 2030, and 700 million by 2045. The global prevalence of diabetes is growing rapidly, especially in the developing world, and the prevalence of diabetic kidney disease (DKD) and end-stage renal disease (ESRD) continues to rise (Hu, 2011). DKD is one of the most feared diabetic chronic microvascular complications and the major cause of ESRD. All-cause mortality in individuals with DKD is approximately 30 times higher than that in diabetic patients without nephropathy. Recent studies indicate that renal tubular epithelial-mesenchymal transition (EMT) and extracytoplasmic matrix (ECM) accumulation in the renal interstitium play key factors in the pathogenesis of DKD (He et al., 2015; Yao et al., 2014). Increasing studies have reported that the progression of renal fibrosis in diabetic nephropathy was mediated by many signaling pathways. TGF-β has been implicated as a key pathogenic factor in most types of CKD associated with fibrosis, including DKD (Lv & Zhang, 2019). The heteromeric signaling complex, resulting from the binding of TGF-β1 to type II receptor and in turn type I receptor, transduces signals through receptor-regulated Smads (Smad2/3) and common-partner Smad (Smad4), leading to transcriptional regulation of target genes (Sierra et al., 2015). ROS accumulation can lead to oxidative stresses in the cell, leading to damage of cellular components including protein and DNA. Ultimately, oxidative stress leads to renal fibrosis and a decline in renal function. NADPH oxidases (NOX) are accepted as major sources of ROS generation in diabetic nephropathy and chronic kidney disease. The up-regulation and the hyper-activation of NOX also likely contribute to oxidative stress in pathophysiologic stages. Elevation of the renal ROS level through hyperglycemia-mediated NOX activation results in oxidative stress, which induces damage to kidney tissues, causing DKD (Lee, An, Kim, & Bae, 2020).
Metabolomics is a new and important omics technology for qualitative and quantitative analysis of all small molecular metabolites in living cells, tissues, and body fluids. As a new discipline to identify the overall metabolic changes of living organisms, metabolomics provides new insights into the study of diabetes and diabetic complications (Lu, Xie, Jia, & Jia, 2013).
The purpose of this study was to investigate the protective effect of natural product acteoside on DKD of diabetic male db/db mice and HK-2 cells induced by high glucose in vitro and its mechanism. So, in this study, db/db mice were adopted to evaluate the renal protective effects of acteoside, and the regulation of different metabolites in serum was analyzed to illuminate the mechanisms of acteoside. Moreover, the high glucose-induced HK-2 in vitro model evaluates the efficacy of acteoside on db/db mice by determination of NADPH/ oxidase-TGF-β/Smad signaling pathway expression levels.
2 | MATERIALS AND METHODS
2.1 | Chemicals and instruments
UPLC-grade acetonitrile was purchased from Merck (Darmstadt, Germany); formic acid was purchased from Sigma-Aldrich (Sigma, St. Louis, Missouri). Acteoside (Purity ≥ 98%) was purchased from the National Institutes for Food and Drug Control (Beijing, China). The structure of the acteoside is shown in Figure 1A. Serum urea nitrogen (BUN) reagent kit, aspartate aminotransferase (GOT) reagent kit, alanine aminotransferase (GPT) reagent kit, total cholesterol (TCHO) reagent kit, triglyceride (TG) reagent kit, serum creatinine (Scr) reagent kit, urinary microalbumin (mALB) reagent kit, and insulin (INS) reagent kit were bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Metformin hydrochloride tablets were purchased from Sino American Shanghai Squib Pharmaceutical Ltd; Irbesartan was purchased from Shenzhen Haibin Pharmaceutical Co., Ltd. DMEM and F12 were purchased from GIBCO, America; NOX1 (catalog number: ab121009), NOX2 (catalog number: ab129068), NOX4 (catalog number: ab154244), and Smad7 (catalog number: ab216428) first antibody was purchased from Abcam (Cambridge, United Kingdom). α-SMA (catalogue number: 19245), E-cadherin (catalogue number:3195), NF-κB p65 (catalogue number: 8242), TGF-β1 (catalogue number: 3711), Smad2 (catalogue number: 5339), Smad3 (catalogue number: 9523), Smad4 (catalogue number: 46535), and PSmad2/3(catalogue number: 8828) first antibody were purchased from CST. All other chemicals and reagents used in this study were of analytical grade and made in China.
Waters Acquity™ Ultra Performance LC system (Waters) equipped with a Quattro Micro MS spectrometer and a Waters Xevo TM G2 QTof MS (Waters MS Technologies, Manchester, New Hampshire). Deionized water was purified on a Milli-Q system (Millipore, Bedford, Massachusetts). Mass Lynx v4.1 workstation was adopted to analyze the data, and an ultra-high-speed centrifuge at low temperature (Thermo Scientific, United Kingdom) and DMI3000M microscope (Leica, Germany) were used.
2.2 | Animal experimental conditions
All the experimental procedures and protocols used in this study were reviewed and approved by the Institutional Animal Ethics Committee of Nanjing University of Chinese Medicine (Nanjing, China). The protocols described in the present study were in agreement with the institutional guidelines for the care and use of laboratory animals. The mice were purchased from the Animal Model Research Center of Nanjing University (Certificate no. Su 2015–0001). All experiments were performed with 8-week-old male db/db mice and age-matched wild-type db/m littermates mice with an initial body weight of 30 40 g. The db/db diabetic mouse model is an internationally recognized animal model of diabetes. Studies have shown that the mechanism of DKD in db/db mice is similar to that of humans. Animals were housed in cages with a constant humidity (ca. 60% ± 2%) and temperature (ca. 23 ± 2℃) and with a light/dark cycle of 12 hr. The animals were undergoing an adaptation period of 3 weeks, during which they were allowed unlimited access to chow and tap water. After the acclimation period, the db/db mice were randomly divided into four groups with six in each group: model group (normal saline, M), metformin group (250 mg kg-1 d-1, EJSG), irbesartan group (50 mg kg-1 d-1, EBST), acteoside group (70 mg kg-1 d-1, MRHTG), once a day (Gao, Peng, Huo, Liu, & Yan, 2015). Moreover, in this study, metformin and irbesartan were selected as positive control drugs. It has been reported that irbesartan can decrease blood pressure, blood lipid, and kidney lipid. It has no effects on blood glucose and liver lipid. It can improve the function and pathological change of kidneys of db/db mice. The protective effect of metformin on the kidney is reflected in the reduction of proteinuria in diabetic rats and patients with type 2 diabetes. The animal dose of metformin and irbesartan was extrapolated from the human daily dose, using the body surface area normalization method. Then, the mice were given the corresponding drug once a day by gastric lavage for 6 weeks, and the db/m mice were used as the control group, which were given the same volume of saline at the same time.
2.3 | Samples collection
During the experimental period, body weights and fasting blood glucose (FBG) levels were recorded weekly, FBG levels were measured by a One Touch Ultra II blood glucose monitoring system (Life Scan, Milpitas, California) by tail vein every Monday (at 8 a.m.). After 6 weeks of administration, the mice were fasted in metabolic cages with free access to water to collect 24 hr urine. All urine samples were immediately centrifugated at 3000 rpm for 10 min after collection, and the supernatants were separated and stored at -80℃ until analysis. At the end of the study, mice were sacrificed under anesthesia with 10% chloral hydrate (3 mg/kg), and blood was collected for the concentration of biochemical parameters and metabolomics study. Then, the blood samples were centrifugated at 3000 rpm for 10 min, and the serum samples were separated and stored at -80℃ until analysis. The right kidney was removed with one piece fixed with 10% neutral buffered formalin for histologic examination. The renal cortex was isolated from the other piece and frozen in liquid nitrogen for western blotting.
Cistanche for treating renal failure
2.4 | Cell culture and treatment
The human RTEC line HK-2 was obtained from Nanjing Keygen Biotechnology Development Co., Ltd. The complete medium was low glucose DMEM with 10% FBS and 1% penicillin-streptomycin solution. The HK-2 cells were cultured in 37℃, 5% CO2, and saturated humidity. The cells were subcultured at 80% confluence, which was removed from the incubator, and the original medium in the dish was discarded. Cells were rinsed using 0.5 ml phosphate buffer saline (PBS) and digested by treatment with 0.5 ml trypsin for 1–2 min at 37℃. Digestion was terminated using a 2 ml DMEM medium, and the cell suspension was subsequently centrifuged at 450g for 5 min at 4C. The supernatant was discarded and cells were resuspended in 2 ml corresponding DMEM medium to obtain a single-cell suspension. Cells were seeded into different dishes/microplates at different densities for subsequent experimentation, as described below. HK-2 cells were plated on 96-well plates and dishes, pretreated with DMEM low glucose (5 mmol/L), incubated at 37C and 5% CO2 for 6 hr, and then were synchronized. The experimental groups were divided as follows: control group (C), without intervention factor; model group (M), in which cells were cultured in high glucose (30 mmol/L) DMEM solution; osmotic pressure control group (DMEM+24.5 mmol/L mannitol, GLC); irbesartan group (25 mmol/L) and acteoside (MRHTG) group at five dosages (5, 10, 25, 50, and 100 μmol/L), respectively.
2.5 | Cell morphology
The HK-2 cells were washed twice with PBS and viewed under an inverted microscope (OLYMPUS IX51). The morphology of the cells was observed under 100 magnifications.
2.6 | Biochemical indicators measurements
The therapeutic efficacy of acteoside on db/db mice was evaluated for the levels of mALB in urine and FBG, INS, T-CHO, TG, Scr, GOT, GPT, and BUN in serum, which were detected followed by the description supplied by the kit's manufacturer.
2.7 | Pathological analysis
Part of the renal cortex was performed for hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining in order to observe pathological changes in renal tissue, degrees of fibrosis tissue hyperplasia, structures of glomeruli and tubules through an electron microscope (400). When taking a photograph, try to fill the whole field of vision with kidney tissue to ensure that the background light of each photograph is the same. Image-Pro Plus 6.0 software was used to select the same red-purple color as the unified criterion for judging the positive of all photos. The accumulated optical density (IOD) of basement membrane positive expression and the pixel area of glomerular vascular plexus (AREA) were obtained by analyzing each photo, and the average optical density (IOD/AREA) was calculated.
2.8 | Western blotting
Kidney tissue and cells were extracted with lysis buffer; the lysates were centrifuged. After full pyrolysis, centrifuge for 5 mins at 12000 rpm, and take the supernatant and the supernatants were collected. Bicinchoninic acid (BCA) protein concentration kit was used to determine protein concentration. After quantification, the protein was resolved by 10% SDS-PAGE, transferred onto PVDF membranes, then blocked in 5% BSA in TBST, and incubated with primary antibodies followed by the corresponding secondary antibodies. The antibody reactivity was then detected by ECL and quantified with ImageJ software. Renal tissues samples were analyzed by western blotting for detecting the expression levels of α-SMA, TGF-β1 (1:1300), Smad2 (1:3000), Smad3 (1:1000), P-Smad2/3 (1:500), Smad4 (1:1000), and Smad7 (1:1000) protein.
2.9 | ELISA analysis
The secretion of monocyte chemoattractant protein -1 (MCP-1), interleukin –1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in the supernatant of each cell were measured by ELISA kit.
2.10 | Real-time PCR
Moreover, these cells were prepared for the real-time PCR to examine the expression levels of NOX1, NOX2, NOX4, α-SMA, E-cadherin, NF-κB p65, TGF-β1, Smad2, Smad3, Smad4, and Smad7 mRNA. Cells in each group (about 1 107 cells) were collected. Total RNA was extracted according to Trizol's one-step method, and its purity and concentration were determined. With GAPDH as internal reference, the primer sequence was the following. TGF-β1, sense primer: 5-ACACATCAGAGCTCCGAGAA-3, antisense primer: 5-GAGGTATC GCCAGGAATTGT-3;E-cadherin, sense primer: 5-ACGCATTGCCA CATACACTC-3, antisense primer: 5-GGTGAATTCGGGCTTGTTGT-3; NF-κB p65, sense primer: 5-CTTCCTGCCCTACAGAGGTC-3, antisense primer: 5-AGAGCAAGGAAGTCCCAGAC-3;NOX1, sense primer: 5-CCTAGAAGGGCTCCAAACCA-3, antisense primer: 5-GGAAGGCATCCACAAACAGG-3; NOX2, sense primer: 5-ACCCTTCGCATCCATTCTCA-3, antisense primer: 5-TCTGCAAACCACTCAAAGGC-3; NOX4, sense primer: 5-CAAGCAGGAGAACCAGGAGA-3, antisense primer: 5-AGTTGAGGGCATTCACCAGA-3; Smad2, sense primer: 5-TGAGCACGTGAGGTGAGATT-3, antisense primer: 5-CTCCATCACAGTGCACCAAG-3; Smad3, sense primer: 5-CAGCCGGTTTGGATTACAGG-3, antisense primer: 5-GAGTCAAAGTCCCTGCTCCT-3; Smad4, sense primer: 5-CACTGCCAACTTTCCCAACA-3, antisense primer: 5-ATCCATTCTGCTGCTGTCCT-3; Smad7, sense primer: 5-AAGAGTCAGCTGGTGCAGAA-3, antisense primer: 5-GCGGACTTGATGAAGATGGG-3; α-SMA, sense primer: 5-GGTGCTGTCTCTCTATGCCT-3, antisense primer: 5-CAGATCCAGACGCATGATGG-3; GAPDH, sense primer: 5-TGGTATCGTGGAAGGACTCA-3, antisense primer: 5-CCAGTAGAGGCAGGGATGAT-3.
2.11 | Statistical analysis
The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology. All experiments were randomized and blinded. The results are expressed as the M ± SD/SEM of the indicated number (n) of independent experiments. Data in the current study are presented as the M ± SD/SEM from at least three independent experiments. The significance of differences was analyzed by one-way ANOVA followed by Tukey's test (more than two groups) or a Student's t-test (two groups). The post hoc tests are performed only when F achieved p < .05, and there was no significant variance inhomogeneity. All tests were two-sided, and p < .05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 7.0 software.
Cistanche acteoside for improving Kidney
2.12 | UPLC-QTOF/MS conditions and data analysis
All serum samples were thawed at room temperature before preparation. The 300 μl acetonitrile was added to 100 μl serum samples to precipitated protein, and vigorously mixed for 60 s. All the samples were centrifuged at 13000 rpm for 10 mins at 4℃. Finally, the supernatant was injected into UPLC-QTOF/MS analysis. Metabolite separation was performed using a Waters Acquity™ Ultra Performance LC system (Waters) equipped with a Waters Xevo™ G2 Q/TOF-MS (Waters MS Technologies). An aliquot of 2 μl of sample solution was injected on an Acquity UPLC BEH C18 (100 mm 2.1 mm, 1.7 μm, Waters Corporation, Milford, Connecticut) at 35℃ and the flow rate was 0.4 ml/min. The optimal mobile phase consisted of water (A) (containing 0.1% formic acid) and acetonitrile (B). The optimized UPLC elution conditions for serum analysis were 0 3 min, 95% 55% A; 4 13 min, 55% 5% A; 13 14 min, 5% A. The autosampler was maintained at 4℃.
Mass spectrometry was performed using a Xevo™ G2 QTof (Waters MS Technologies), a quadrupole, and an orthogonal acceleration time-of-flight tandem mass trometer. Leucine-enkephalin was used as the lock mass generating an [M + H]+ ion (m/z 556.2771) and [M-H]-ion (m/z 554.2615) in positive and negative modes, respectively. The concentration of leucine-enkephalin was 200 pg/ml and the infusion flow rate was 100 μl/min to ensure accuracy during the MS analysis via a syringe pump. Data were collected in centroid mode from 100 to 1,000 m/z. For both positive and negative electrospray modes, the capillary and cone voltage was set at 3.0 kV and 30 V, respectively. The desolvation gas was set to 600 L/hr at a temperature of 350℃, the cone gas was set to 50 L/hr and the source temperature was set to 120℃. The data acquisition rate was set to 30 ms, with a 0.02 s interscan delay.
In addition, 10 serum samples were randomly selected from each group and mixed together as the quality control (QC) samples, respectively. The QC sample was used to optimize the condition of UPLCQTOF/MS, as it contained most information of the whole sample. The QC samples were injected six times at the beginning of the running in order to condition or equilibrate the system and then every 10 samples to further monitor the stability of the analysis. Every day, after the instrument was calibrated, the QC sample was firstly analyzed to test the stability of the instrument in order to ensure the consistent performance of the system.
All of the data acquisition and analyses of data were controlled by Waters MassLynx v4.1 software. The multivariate data matrix was analyzed by EZinfo software 2.0 (Waters Corp.). The main parameters include retention time range 0 14 min; mass ratio m/z 100 1,000, mass tolerance range 0.01 Da, peak intensity threshold 50, quality window 0.05 Da, retention time windows 0.20 min, and automatic detection of 5% peak height and noise. The intensity of each ion was normalized with respect to the total ion count to generate a data matrix that consisted of the retention time, m/z value, and the normalized peak area.
The resultant data matrices were introduced to EZinfo 2.0 software for principal component analysis, partial least-squares discriminant analysis (PLS-DA), and orthogonal projection to latent structures (OPLS-DA) analysis. From the OPLS-DA, various metabolites could be identified as being responsible for the separation between the control group and model group and were therefore viewed as potential markers. Potential markers of interest were extracted from S-plots constructed following analysis with OPLS-DA, and variables that had significant contributions to discrimination between groups were subjected to further identification of the molecular formula.
The variable importance (VIP) in the projection value is a weighted sum of squares of the PLS weights, and the variables with VIP > 1 were considered to be influential for the separation of samples in the score plots generated from OPLS-DA analysis. In all experiments, the confidence level was set at 95% to determine the significance of the difference (p < .05). Those variables were eventually selected as potential biomarkers. The PLS-DA score plots were described by the cross-validation parameter R2Y and Q2, which represents the total explained variation for the X matrix and the predictability of the model, respectively. Excellent models are obtained when the cumulative values of R2Y and Q2 are above 0.8. The relative distances between administration groups and the control group from the PLS-DA score plot were calculated with the average value (x-axis and y-axis) of all samples of the control group as the referenced point.
Potential metabolites selected were identified according to the determination of the accurate m/z, retention time, and typical MS/MS fragment and pattern of the potential biomarkers above, which were obtained in the positive and negative ion modes. The construction of the metabolic pathway was performed with Metabo Analyst, which is a web-based tool for visualization of metabolomics (https://www. metaboanalyst.ca/) based on database sources including the KEGG (HTTP:// www.genome.jp/kegg/) and the HMDB (http://www.hmdb. ca/) databases for searching. The retention time and typical MS/MS fragment and pattern had great avail to narrow the range of possible molecules.
Acteoside in Cistanche can Improve Kidney Function
3 | RESULTS
Click here for information about Part II (results) of this article.
Excerpted from: ' A natural product of acteoside ameliorate kidney injury in diabetes db/db mice and HK-2 cells via regulating NADPH/ oxidase-TGF-β/Smad signaling pathway' -----by Qinwen Wang | Xinxin Dai et.al
----Phytotherapy Research. 2021;35:5227–5240. wileyonlinelibrary.com/journal/ptr © 2021 John Wiley & Sons Ltd. DOI: 10.1002/ptr.7196
